Methods of Treatment

ABSTRACT

The present disclosure provides a biochemical basis of nephrotic syndrome and provides and explanation for the observed proteinuria and other effects. As a result, the present disclosure provides method for treating and/or preventing nephrotic syndrome as well as methods of alleviating symptoms associated with nephrotic syndrome. The present disclosure further provides methods for reducing proteinuria in nephrotic syndrome and other disease states as discussed herein.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 14/943,167, filed Nov. 17, 2016, which is currently pending. U.S.application Ser. No. 14/943,167 is a continuation of U.S. applicationSer. No. 13/152,169, filed on Jun. 2, 2011, now abandoned. U.S.application Ser. No. 13/152,169 claims the benefit of and priority toU.S. provisional application 61/351,865, filed on Jun. 5, 2010, nowexpired.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

Funding for the work described herein was provided by the federalgovernment, which has certain rights in the invention.

FIELD OF THE DISCLOSURE

The present disclosure is directed to methods for the treatment andprevention of nephrotic syndrome, diabetic conditions and conditionsrelated thereto.

BACKGROUND

Nephrotic syndrome is caused by various disorders that damage thekidneys, particularly specialized cells called podocytes and thebasement membrane of the glomerulus. Diabetic nephropathy and membranousglomerulonephritis (also called membranous nephropathy) are commoncauses in adults, whereas minimal change disease is the most commoncause in children. Characteristics of nephrotic syndrome includes lossof protein in the urine (proteinuria), hyperlipidemia(hypercholesterolemia and hypertriglyceridemia), hypoalbuminemia (lowblood albumin or protein levels) and edema. Proteinuria is defined asthe presence of an excess of serum proteins in the urine. Albuminuria, aspecific type of proteinuria, is a pathological condition whereinalbumin is present in the urine.

Podocytes (or visceral epithelial cells) are cells in the glomerularcapillary loop in the kidneys. The glomerulus filters blood, holdingback large molecules such as proteins, and passing through smallmolecules such as water, salts, and sugar, as the first step in formingurine. The long processes, or “foot projections,” of the podocytes wraparound the capillaries, and leave slits between them. Blood is filteredthrough these slits. Kidneys affected by nephrotic syndrome have smallpores in the podocytes which are large enough to permit proteins totransit, causing proteinuria.

When protein is lost in the urine, its blood concentration decreases,allowing water to move into other areas of the body, which leads toswelling known as edema. Edema is commonly observed in the feet andlegs, in the belly or abdomen (ascites), and around the eyes, but canoccur anywhere, especially in response to gravity. Additionally, becauseof this extra fluid that stays in the body, people often gain weight,experience fatigue and may find that they urinate less often.

Many conditions are categorized as nephrotic syndromes, includingminimal change disease (MCD), focal segmental glomerulosclerosis (FSGS),membranous nephropathy (MN) (also called membranous glomerulonephritis,MGN), and membranoproliferative glomerulonephritis (MPGN). For yearspathologists found no changes in MCD tissue when viewing specimens underlight microscopy, hence the name minimal change disease. With the adventof electron microscopy, the changes now known as the hallmarks for thedisease include diffuse loss of podocyte foot processes, vacuolation ofthe podocyte foot processes, and growth of microvilli on the visceralepithelial cells. Diabetic nephropathy is the most common cause ofnephrotic syndrome in developed countries.

Hypertriglyceridemia may occur due to changes in the activity of enzymesthat degrade triglycerides, such as lipoprotein lipase (2-4).

The molecular basis of nephrotic syndrome is not known. Furthermore, theassociation of proteinuria and glucocorticoid sensitivity in nephroticsyndrome and the link between proteinuria and hypertriglyceridemia, twokey components of nephrotic syndrome, have yet to be established.Therapy designed to reduce proteinuria further complicates the study ofdisease mechanisms. For example, glucocorticoids used to treatproteinuria in MCD independently raise plasma triglyceride levels (5),and normalization of plasma triglyceride levels lags behind the responseof proteinuria to glucocorticoids in certain forms of nephroticsyndrome, such as MCD (6).

The present disclosure provides a disclosure of the biochemical basis ofnephrotic syndrome (exemplified by a model of MCD) and provides anexplanation for the observed proteinuria and other effects. As a result,the present disclosure provides method for treating and/or preventingnephrotic syndrome, such as but not limited to, diabetic nephropathy,MCD, FSGS, MN/MGN, and MPGN, as well as methods of alleviating symptomsassociated with nephrotic syndrome, including, but not limited to,proteinuria, hypercholesterolemia, hypertriglyceridemia, hypoalbuminemiaand edema. The present disclosure further provides methods for treatingand preventing diabetic conditions and physiological effects thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1I show Angptl4 mRNA and protein expression in experimental andhuman MCD.

FIG. 1A shows induction of nephrotoxic serum (NTS) induced heterologousphase proteinuria (n=4 rats/group); *** P<0.001.

FIG. 1B shows glomerular Angptl4 mRNA expression was upregulated duringnephrotoxic serum (NTS) induced heterologous phase proteinuria shown inFIG. 1A (n=4 rats/group); *** P<0.001.

FIG. 1C shows NTS injected Angptl4 −/− mice (n=4 mice/group) developsignificantly lower albuminuria, indicating a role of Angptl4 in thedisease process; * P<0.05.

FIG. 1D shows NTS injected Angptl4 −/− mice (n=4 mice/group) developedonly 30% foot process effacement compared to diffuse effacement inAngptl4 +/+ mice.

FIG. 1E shows by confocal imaging that Angptl4 in normal rat glomerulico-localized with podocyte protein CD2AP, indicating expression inpodocytes. Absorbing out reactivity from anti-Angptl4 antibody withrecombinant Angptl4 abolished immunoreactivity.

FIG. 1F shows significant glomerular mRNA upregulation of Angptl4 wasseen after injection of a single dose of puromycin aminonucleoside (PANmodel) starting on Day 3 (peak up to 80 fold increase in differentstudies). Lesser upregulation developed in PHN, and no significantchange was detected in anti-Thy1.1 nephritis or a rat model of non-HIVcollapsing focal and segmental glomerulosclerosis (CG) using sievedglomeruli or glomeruli isolated by laser capture microdissection (LCM)[threshold for significance is 3-fold change (7)].

FIG. 1G shows glomeruli had increased Angptl4 expression (red) thatoverlapped partially (white arrows) with GBM heparan sulfateproteoglycans (white, intensity reduced in PAN) and podocyte nephrin(green) on day 6 after injection of a single dose of puromycinaminonucleoside (PAN model).

FIG. 1H shows Immunogold EM of PAN Model day 6 kidney demonstrating goldparticles in the podocyte (yellow arrows) and GBM (black arrows);magnification: EM 40,000×.

FIG. 1I shows kidney biopsies from MCD patients (n=5 patients) revealedincreased glomerular expression of Angptl4 (red) that overlapssubstantially with nephrin and GBM laminin, and only marginally withendothelial PECAM1 staining; magnification: light microscopy 400×.

FIGS. 2A-F show the generation and characterization of male Angptl4transgenic rats.

FIG. 2A show a transgenic (TG) rat model for podocyte specific overexpression of Angptl4 in podocytes.

FIG. 2B shows tissue specific over expression of Angptl4 mRNA (n=3rats/group) consistent with expression in podocytes; *** P<0.001.

FIG. 2C shows PAS stained sections from 3 month old heterozygous TG rats(n=3 rats/group) and revealed normal morphology (magnification 400×),with prominent podocytes in NPHS2-Angptl4 TG rats (arrows).

FIG. 2D shows confocal imaging and revealed increased glomerularexpression of Angptl4 protein (magnification 400×) that overlapped withpodocyte nephrin (magnification 630×) and GBM laminin (magnification630×) in NPHS2-Angptl4 TG rats (n=3 rats/group).

FIG. 2E shows that by age 5 months, diffuse foot process effacement wasnoted on EM in homozygous NPHS2-Angptl4 TG rats.

FIG. 2F shows immunogold EM analysis of these rats and revealed aprogression from intact foot processes (FP) containing gold particleclusters and scattered GBM particles at age 1 month, to partialeffacement with GBM gold particle clusters opposite to effaced footprocesses (EFP) reaching up to the endothelial (ENDO) surface, andfinally diffuse effacement with dense gold particle clusters in the GBMby age 5 months. Electron microscopy magnification 15,000×-40,000×.

FIGS. 3A-H show the relationship of An t14 overexpression withproteinuria.

FIG. 3A shows female homozygous NPHS2-Angptl4 TG rats developedsignificant albuminuria (n=6 rats/group); * P<0.05; ** P<0.01, ***P<0.001.

FIG. 3B shows male heterozygous NPHS2-Angptl4 TG rats developedsignificant albuminuria (n=6 rats/group); * P<0.05; ** P<0.01, ***P<0.001.

FIG. 3C shows male homozygous NPHS2-Angptl4 TG rats developedsignificant albuminuria (n=6 rats/group); ** P<0.01.

FIG. 3D shows GelCode blue stained SDS PAGE assessment of urinaryprotein (3 μg/lane, except MCD remission) followed by densitometry ofthe bands (n=3 readings/value) and shows a predominance of albuminuria(intact albumin band at 70 kDa, arrow) in NPHS2-Angptl4 TG rats similarto human MCD.

FIG. 3E shows immunogold EM with anti-V5 antibody to specifically detecttransgenic protein in 3 month heterozygous TG male rats and showed goldparticles in effaced foot processes (EFP) and GBM in NPHS2-Angptl4 TGrats; EM 40,000×.

FIG. 3F shows induction of PAN in heterozygous male NPHS2-Angptl4 (lowdose) worsened, proteinuria compared to wild type littermates (n=8rats/group); * P<0.05; ** P<0.01, *** P<0.001.

FIG. 3G shows rats treated with glucocorticoids (PAN-S) on alternatedays starting 1 day after induction of PAN showed transient reduction inDay 6 proteinuria (n=4 rats/group); * P<0.05; ** P<0.01,

FIG. 3H is a study of glomerular gene expression from the study in 3G,and shows that Angptl4 is a glucocorticoid sensitive gene. * P<0.05; **P<0.01

FIGS. 4A-D show the relationship of Angptl4 sialylation to proteinuria.FIG. 4A shows 2D gel electrophoresis (200 μg protein/gel) and Westernblot of protein from perfused glomeruli (upper panel, control; middlepanel PAN; lower panel PAN plus the addition of glucocorticoid). In thecontrol panel, analysis revealed the presence of small amounts offragments (red arrow; 1) and monomers (yellow arrow; 2) of Angptl4, andlarger amounts of low order oligomers (pink arrows; 3) of Angptl4migrating at neutral or high pI. On PAN Day 6 (middle panel), high andneutral pI oligomeric forms were increased (pink arrow, 3; orange arrow4), whereas treatment with glucocorticoids (see FIG. 3G) blunts thisincrease (lower panel). Of the Angptl4 migrating at neutral pI,fragments were reactive with anti-phosphothreonine antibodies, whereasoligomers are reactive with sialic acid binding lectin MAA (exemplifiedfor PAN, excerpts from independent blots).

FIG. 4B shows densitometry analysis of the Western blot from FIG. 4A; **P<0.01; * * * P<0.001.

FIG. 4C shows 2D gel electrophoresis (150 rig protein/gel) and Westernblot of proteins from Angptl4-HEK293 stable cell line incubated withcontrol and probed with antibodies to MAA lecithin (top panel), antiAngplt4 antibodies (upper middle panel); Angptl4-HEK293 stable cell lineincubated with sialic acid precursor ManNAc (25 mM) and probed withanti-Angptl4 antibodies (lower middle panel) and Angptl4-HEK293 stablecell line incubated with sialic acid precursor ManNAc (25 mM) and probedwith antibodies to MAA lecithin (lower panel). Analysis of theconcentrated supernatant revealed a shift from high pI forms (greenarrow and line in upper middle panel) towards neutral pI forms (bluearrow in lower middle panel and lower panel), that were MAA reactive.

FIG. 4D shows that feeding NPHS2-Angptl4 TG rats with 1 mg/ml ManNAc intap water for 12 days (d) caused a significant reduction in 18 houralbuminuria (nadir on Day 12 was 59.4±3.3% of baseline), which returnedtowards baseline values after 12 days of washout, and approacheduntreated control NPHS2-Angptl4 TG rat values on day 24 of washout(individual tracings and pilot study data in FIG. 6). In panel d, all *differences are with baseline values. * P<0.05; ** P<0.01; *** P<0.001.

FIGS. 5A-C show characterization of recombinant Angptl4 produced by aHEK293 stable cell line.

FIG. 5A shows HEK293 cells stably transfected with a pcDNA-ratAngptl4-V5-His expression construct line showed 55,000 fold upregulationof Angptl4 mRNA expression compared to a control empty vector cell line.*** P<0.001

FIG. 5B shows Angptl4-HEK293 cells secreted mostly intact protein intothe supernatant under serum free conditions (as demonstrated by 2D gelelectrophoresis and Western blot with pre-immune serum and anti-Angptl4antibodies).

FIG. 5C shows 2D gel electrophoresis of 200 ug human plasma (n=4patients/group) and demonstrates that only in patients with MCD relapsewas elevated circulating levels of the 55-70 kDa pI 8-8.5 Angptl4protein observed (oval in MCD relapse panel). Increased circulatinglevels of neutral PI monomers and oligomers were observed in MCDpatients in relapse (arrow in in MCD relapse panel) and monomers only inpatients with MN (arrow in MN panel).

FIGS. 6A-D show administration of ManNAc reduced albuminuria in a ratmodel.

FIG. 6A shows representative tracing of albuminuria from pilot studieswith heavily albuminuric NPHS2-Angptl4 TG rats receiving increasingdoses of ManNAc (1 to 8 mg/ml). The study revealed that ManNAc reducedalbuminuria in these animals.

FIG. 6B shows individual tracings from the study group of NPHS2-Angptl4TG rats treated with ManNAc (1 mg/ml) that completed the study. Twourine collections 12 days apart were conducted prior to the start of thestudy to ensure that these rats developed increasing albuminuria withtime. The study revealed that ManNAc (1 mg/ml) reduced albuminuria inthese animals.

FIG. 6C shows 2D electrophoresis and Western blotting (200 μgprotein/gel) of glomeruli from Day 12 ManNAc or control TG rats usinganti-Angptl4 antibodies or lectin SNA I (n=3 blots/ condition).Reproducible neutral pI Angptl4 spots are bordered by red oval (1), andhigh pI by green oval (2). Neutral pI spots are also reactive withsialic acid binding lectin SNA I.

FIG. 6D shows densitometry of Angptl4 spots of FIG. 6C and indicates asignificant increase in the percentage of neutral pI forms in ManNAC fedrats. *** P<0.001.

FIGS. 7A-D show improvement in nephrotic syndrome parameters in ManNActreated rats with puromycin aminonucleoside nephrosis (PAN model). InFIGS. 7A-7D, rats (n=5 rats/group) received a single intravenousinjection of puromycin aminonucleoside (10 mg/100 gm), and were treatedwith ManNAC 80 mg/Kg body weight in tap water starting on Day 4, whichcoincides with the onset of proteinuria.

FIG. 7A shows a significant reduction in proteinuria in ManNAc treatedrats on day 6 post administration of puromycin aminonucleoside. *P<0.05; ** P<0.01; *** P<0.001.

FIG. 7B shows a significant improvement in plasma albumin levels inManNAc treated rats on day 6 post administration of puromycinaminonucleoside. * P<0.05; ** P<0.01; * * * P<0.001.

FIG. 7C shows a significant reduction in hypercholesterolemia in ManNActreated rats on day 6 post administration of puromycinaminonucleoside. * P<0.05; ** P<0.01; *** P<0.001.

FIG. 7D shows a significant reduction in hypertriglyceridemia in ManNActreated rats on day 6 post administration of puromycinaminonucleoside. * P<0.05; ** P<0.01; *** P<0.001.

FIGS. 8A-C show the effect of ManNAc therapy on proteinuria in diabeticanimals

FIG. 8A shows 2D gel electrophoresis and Western blot analysis ofglomeruli from diabetic db/db and control db/m mice using ananti-Angptl4 Ab. The results show increased expression of Angptl4 indiabetic mouse glomeruli as compared to control, including the high p1forms that are involved in the pathogenesis of proteinuria.

FIG. 8B shows 2D gel electrophoresis and Western blot analysis ofglomeruli from Zucker Diabetic Fatty rats and control normoglycemic ratsusing an anti-Angptl4 Ab. The results show increased expression ofAngptl4 in rat glomeruli from Zucker Diabetic Fatty rats as compared tocontrol, including the high pI forms that are involved in thepathogenesis of proteinuria.

FIG. 8C shows ManNac treatment decreased proteinuria in Zucker DiabeticFatty rats. 13 week old male Zucker Diabetic Fatty rats were treatedwith ManNAc in tap water or with plain tap water (n=4 rats/group) at 70and 140 mg/Kg. Significant reduction in proteinuria was noted in theManNAc treated group. * P<0.05; ** P<0.01.

FIGS. 9A-B show the amino acid and cDNA sequences of Angptl4 fromvarious species. SEQ ID NOS. 1 and 2 show amino acid and cDNA sequencefrom human (Protein Variant 1 isoform a, long form; underlined aminoacid sequences at a position 40 and 161-164); SEQ ID NOS. 3 and 4 showamino acid and cDNA sequence from human (Protein Variant 3 isoform b,short form; underlined amino acid sequences at a position 40 and161-164); SEQ ID NOS. 5 and 6 show amino acid and cDNA sequence fromrat; SEQ ID NOS: 7 and 8 show amino acid and cDNA from mouse.

SUMMARY OF THE DISCLOSURE

In a first aspect, the present disclosure provides methods of treatmentand/or prevention of nephrotic syndrome in a subject. In one embodiment,the nephrotic syndrome is characterized as minimal change disease (MCD),focal segmental glomerulosclerosis (FSGS), membranous nephropathy(MN)/membranous glomerulonephritis (MGN) membranoproliferativeglomerulonephritis (MPGN), and diabetic nephropathy. In anotherembodiment, the nephrotic syndrome is characterized as MCD. The methodscomprise the step of administering to a subject sialic acid or a sialicacid precursor. The sialic acid or sialic acid precursor may beadministered at a therapeutically effective dose, either alone, as apart of a pharmaceutical composition or in combination with a secondaryagent. Such administration restores normal sialylation of a polypeptide,such as, but not limited to Angptl4, involved in the etiology ofnephrotic syndrome, thereby treating or preventing nephrotic syndrome inthe subject.

In a second aspect, the present disclosure provides methods of treatmentand/or prevention of MCD in a subject. The methods comprise the step ofadministering to a subject a sialic acid or sialic acid precursor. Thesialic acid or sialic acid precursor may be administered at atherapeutically effective dose, either alone, as a part of apharmaceutical composition or in combination with a secondary agent.Such administration restores normal sialylation of a polypeptide, suchas, but not limited to Angptl4, involved in the etiology of nephroticsyndrome, thereby treating or preventing nephrotic syndrome in thesubject.

In a third aspect, the present disclosure provides methods ofalleviating one or more symptoms of nephrotic syndrome, such as, but notlimited to, proteinuria, hypercholesterolemia, hypertriglyceridemia andedema. In one embodiment, the nephrotic syndrome is characterized asMCD, FSGS, MN/MGN, MPGN or diabetic nephropathy. In another embodiment,the nephrotic syndrome is characterized as MCD. The methods comprise thestep of administering to a subject sialic acid or a sialic acidprecursor. The sialic acid or sialic acid precursor may be administeredat a therapeutically effective dose, either alone, as a part of apharmaceutical composition or in combination with a secondary agent.Such administration restores normal sialylation of a polypeptide, suchas, but not limited to Angptl4, involved in the etiology of nephroticsyndrome, thereby alleviating one or more symptoms of nephrotic syndromein the subject.

In a fourth aspect, the present disclosure provides methods for reducingproteinuria in a subject. In one embodiment, the subject is sufferingfrom nephrotic syndrome. In one embodiment, the nephrotic syndrome ischaracterized as MCD, FSGS MN/MGN, MPGN or diabetic nephropathy. Inanother embodiment, the nephrotic syndrome is characterized as MCD. Inanother embodiment, the subject is suffering from a diabetic condition,such as, but not limited to, diabetic nephropathy, diabetes mellitus,lupus nephritis or primary glomular disease. In a specific embodiment,the proteinuria is caused by hyposialylation of a polypeptide, such as,but not limited to, Angptl4. The methods comprise the step ofadministering to a subject sialic acid or a sialic acid precursor. Thesialic acid or sialic acid precursor may be administered at atherapeutically effective dose, either alone, as a part of apharmaceutical composition or in combination with a secondary agent.Such administration restores normal sialylation of a polypeptide, suchas, but not limited to Angptl4, involved in the induction of proteinuriathereby reducing proteinuria in the subject.

In a fifth aspect, the present disclosure provides methods of reducingedema in a subject. In one embodiment, the subject is suffering fromnephrotic syndrome. In one embodiment, the nephrotic syndrome ischaracterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy. Inanother embodiment, the nephrotic syndrome is characterized as MCD. In aspecific embodiment, the edema is caused by hyposialylation of apolypeptide, such as, but not limited to, Angptl4. The methods comprisethe step of administering to a subject sialic acid or a sialic acidprecursor. The sialic acid or sialic acid precursor may be administeredat a therapeutically effective dose, either alone, as a part of apharmaceutical composition or in combination with a secondary agent.Such administration restores normal sialylation of the polypeptide, suchas, but not limited to Angptl4, involved in the induction of edemathereby reducing edema in the subject.

In a sixth aspect, the present disclosure provides methods of reducinghypercholesterolemia in a subject. In one embodiment, the subject issuffering from nephrotic syndrome. In one embodiment, the nephroticsyndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabeticnephropathy. In another embodiment, the nephrotic syndrome ischaracterized as MCD. In a specific embodiment, the hypercholesterolemiais caused, at least in part, by hyposialylation of a polypeptide, suchas, but not limited to, Angptl4. The methods comprise the step ofadministering to a subject sialic acid or a sialic acid precursor. Thesialic acid or sialic acid precursor may be administered at atherapeutically effective dose, either alone, as a part of apharmaceutical composition or in combination with a secondary agent.Such administration restores normal sialylation of the polypeptide, suchas, but not limited to Angptl4, involved in the induction ofhypercholesterolemia thereby reducing hypercholesterolemia in thesubject.

In a seventh aspect, the present disclosure provides methods of reducinghypertriglyceridemia in a subject. In one embodiment, the subject issuffering from nephrotic syndrome. In one embodiment, the nephroticsyndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabeticnephropathy. In another embodiment, the nephrotic syndrome ischaracterized as MCD. In a specific embodiment, the hypertriglyceridemiais caused by hyposialylation of a polypeptide, such as, but not limitedto, Angptl4. The methods comprise the step of administering to a subjectsialic acid or a sialic acid precursor. The sialic acid or sialic acidprecursor may be administered at a therapeutically effective dose,either alone, as a part of a pharmaceutical composition or incombination with a secondary agent. Such administration restores normalsialylation of the polypeptide, such as, but not limited to Angptl4,involved in the induction of hypertriglyceridemia thereby reducinghypertriglyceridemia in the subject.

In an eight aspect, the present disclosure provides methods of treatmentand/or prevention of diabetic nephropathy, diabetes mellitus, lupusnephritis or primary glomular disease. In a specific embodiment, theforegoing conditions are caused by hyposialylation of a polypeptide,such as, but not limited to, Angptl4. The methods comprise the step ofadministering to a subject sialic acid or a sialic acid precursor. Thesialic acid or sialic acid precursor may be administered at atherapeutically effective dose, either alone, as a part of apharmaceutical composition or in combination with a secondary agent.Such administration restores normal sialylation of the polypeptide, suchas, but not limited to Angptl4, thereby treating or preventing theforegoing conditions in the subject.

In a ninth aspect, the present disclosure provides a composition for usein the methods of the first through eight aspects. The compositioncomprises sialic acid or one or more sialic acid precursors. In oneembodiment, the sialic acid precursor is ManNAc. In an alternateembodiment, the sialic acid precursor is a derivative of ManNAc. Suchcomposition may contain ManNAc and one or more derivatives of ManNAc aswell as a secondary agent.

In a tenth aspect, the present disclosure provides for methods ofdetermining the status of a subject with respect to nephrotic syndromeor a diabetic condition. In one embodiment, the nephrotic syndrome ischaracterized as MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy. Inanother embodiment, the nephrotic syndrome is characterized as MCD. Inone embodiment, the diabetic condition is diabetic nephropathy, diabetesmellitus, lupus nephritis or primary glomular disease. In a specificembodiment, the diabetic condition is diabetic nephropathy. In oneembodiment, such methods determine in the subject the level of apolypeptide associated with nephrotic syndrome or a diabetic condition,such as, but not limited to, Angptl4, the level of sialylation of apolypeptide associated with nephrotic syndrome or a diabetic condition,such as, but not limited to, Angptl4, or a combination of the foregoing.The amount and/or level of sialylation of the polypeptide as determinedfrom the subject may be compared to corresponding amounts and levelsfrom a subject that is diagnosed as not suffering from nephroticsyndrome or a diabetic condition (control subject). Such amounts andlevels may also be compared to a reference standard. A decrease in thelevel of sialylation as compared to the control subject or referencestandard indicates the subject is suffering from or at risk for,nephrotic syndrome or a diabetic condition; the level of sialylation maybe determined with respect to the high pI form of the polypeptide (whichis hyposialylated).

In an eleventh aspect, the present disclosure provides for methods ofdetermining the efficacy of a treatment for nephrotic syndrome or adiabetic condition in a subject undergoing treatment for nephroticsyndrome or a diabetic condition. In one embodiment, the nephroticsyndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabeticnephropathy. In another embodiment, the nephrotic syndrome ischaracterized as MCD. In one embodiment, the diabetic condition isdiabetic nephropathy, diabetes mellitus, lupus nephritis or primaryglomular disease. In a specific embodiment, the diabetic condition isdiabetic nephropathy. The amount and/or level of sialylation of thepolypeptide as determined from the subject during treatment may becompared to corresponding amounts and/or levels from the subject priorto initiating treatment. An increase in the level of sialylation in thesubject undergoing treatment as compared to the level of sialylationdetermined prior to initiating treatment indicates the treatment ishaving the desired effect; the level of sialylation may be determinedwith respect to the high pI form of the polypeptide (which ishyposialylated). Furthermore, the amount and/or level of the polypeptideas determined from the subject during treatment may be compared tocorresponding amounts and/or levels a subject that is diagnosed as notsuffering from nephrotic syndrome or a diabetic condition (controlsubject). Such amounts and levels may also be compared to a referencestandard. A level of sialylation obtained from the subject duringtreatment that is equal to or approaching the level of sialylation fromthe control subject or reference indicates the treatment is having thedesired effect; the level of sialylation may be determined with respectto the high pI form of the polypeptide (which is hyposialylated).

In a twelfth aspect, the present disclosure provides for methods ofidentifying a compound effective for treating or preventing nephroticsyndrome, a diabetic condition or a condition associated therewith. Inone embodiment, the nephrotic syndrome is characterized as MCD, FSGS,MN/MGN, MPGN or diabetic nephropathy. In another embodiment, thenephrotic syndrome is characterized as MCD. In one embodiment, thediabetic condition is diabetic nephropathy, diabetes mellitus, lupusnephritis or primary glomular disease. In a specific embodiment, thediabetic condition is diabetic nephropathy. In general, such screeningmethods comprises the steps of providing an assay system (as describedin more detail below) that expresses a polypeptide involved in theetiology of nephrotic syndrome or a diabetic condition, such as, but notlimited to, Angptl4, introducing into the assay system a test compoundto be tested and determining whether the effect of the test compound onthe level of sialylation of the polypeptide.

DETAILED DESCRIPTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

While investigating the pathogenesis of nephrotic syndrome andproteinuria associated therewith, γ2-nephtotoxic serum (NTS, a serumraised against whole glomeruli), was injected into rats and a panel ofdifferentially expressed glomerular genes was identified (7). Two genesnot previously known to be involved in the pathogenesis of nephroticsyndrome and proteinuria were identified. One of these genes encoded forthe transcriptional factor zinc fingers and homeoboxes 3 (ZHX3)expressed in podocytes, and has now been shown to play a key role in thepathogenesis of MCD (7). The second gene, angiopoietin-like 4, ANGPTL4,was highly upregulated in the podocyte. The expression of the ANGPTL4gene was analyzed in animal models of human glomerular disease,including puromycin nephrosis (PAN), a model of MCD, passive Heymannnephritis (PHN), a model of membranous nephropathy (MN), and anti-Thy1.1nephritis, a model of mesangial injury.

Angiopoietin-like proteins have been implicated in the development ofhypertriglyceridemia and tumor metastasis, and are functionally distinctfrom the angiopoietins. Angptl4 is a PPARγ (8) and PPARα (9) target genehighly expressed in the liver and adipose tissue, strongly induced byfasting in white adipose tissue and liver, and is an apoptosis survivalfactor for vascular endothelial cells under normoxic conditions (10).Angptl4 is a potent inhibitor of LPL (11), inducing significanthypertriglyceridemia following intravenous injection oradenovirus-mediated expression (12, 13). Other studies showed lesserexpression of Angptl4 in cardiomyocytes and skeletal muscle, and lowlevel expression in whole kidney on Northern blot analysis (8). Recentpopulation based studies of the ANGPTL4 gene reveals variants thataffect triglyceride levels in humans (14, 15).

A role of Angptl4 in proteinuria has not been previously reported. Thepresent disclosure shows a conclusive role of podocyte secreted Angptl4in the etiology of in nephrotic syndrome and diabetic conditions. Thepresent disclosure demonstrates for the first time that a large part ofpodocyte secreted Angptl4 in experimental one form of nephrotic syndromeis hyposialylated and that improving sialylation dramatically reducesproteinuria, edema, hypercholesterolemia and hypertriglyceridemia andnormalizes electrophoretic migration of Angptl4. Angptl4 is the firstglucocorticoid sensitive gene to be directly implicated in thepathogenesis of nephrotic syndrome and also the first direct linkbetween proteinuria and hypertriglyceridemia in nephrotic syndrome.Angptl4 amino acid and cDNA sequences from human (Protein Variant 1isoform a, long form and Protein Variant 3 isoform b, short form), ratand mouse are shown in FIG. 8.

Definitions

The terms “prevention”, “prevent”, “preventing”, “suppression”,“suppress” and “suppressing” as used herein refer to a course of action(such as administering a compound or pharmaceutical composition)initiated prior to the onset of a symptom, aspect, or characteristics ofa disease or condition so as to prevent or reduce such symptom, aspect,or characteristics. Such preventing and suppressing need not be absoluteto be useful.

The terms “treatment”, “treat” and “treating” as used herein refers acourse of action (such as administering a compound or pharmaceuticalcomposition) initiated after the onset of a symptom, aspect, orcharacteristics of a disease or condition so as to eliminate or reducesuch symptom, aspect, or characteristics. Such treating need not beabsolute to be useful.

The term “in need of treatment” as used herein refers to a judgment madeby a caregiver that a patient requires or will benefit from treatment.This judgment is made based on a variety of factors that are in therealm of a caregiver's expertise, but that includes the knowledge thatthe patient is ill, or will be ill, as the result of a disease orcondition that is treatable by a method or compound of the disclosure.

The term “in need of prevention” as used herein refers to a judgmentmade by a caregiver that a patient requires or will benefit fromprevention. This judgment is made based on a variety of factors that arein the realm of a caregiver's expertise, but that includes the knowledgethat the patient will be ill or may become ill, as the result of adisease or condition that is preventable by a method or compound of thedisclosure.

The term “individual”, “subject” or “patient” as used herein refers toany animal, including mammals, such as mice, rats, other rodents,rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, andhumans. The term may specify male or female or both, or exclude male orfemale.

The term “therapeutically effective amount” as used herein refers to anamount of a compound, either alone or as a part of a pharmaceuticalcomposition, that is capable of having any detectable, positive effecton any symptom, aspect, or characteristics of a disease or condition.Such effect need not be absolute to be beneficial. When referring tosialic acid or a sialic acid precursor, the term “therapeuticallyeffective amount” refers to an amount of sialic acid or the sialic acidprecursor sufficient to restore normal sialylation patterns to apolypeptide, such as, but not limited to, Angltl4 or an amount of sialicacid or the sialic acid precursor sufficient to reduce proteinuria oranother symptom of nephrotic syndrome in a subject.

The term “pharmaceutically acceptable derivative” means anypharmaceutically acceptable salt, ester, salt of an ester, solvate orother derivative of sialic acid or a sialic acid precursor of thepresent disclosure that, upon administration to a subject, is capable ofproviding (directly or indirectly) sialic acid or a sialic acidprecursor of the disclosure or a metabolite or residue thereof.Particularly favored derivatives are those that increase thebioavailability of sialic acid or the sialic acid precursor of thedisclosure when such are administered to a subject (e.g., by allowing anorally administered compound to be more readily absorbed into theblood), enhance delivery of the sialic acid precursor to a givenbiological compartment, increase solubility to allow administration byinjection, alter metabolism or alter rate of excretion. In oneembodiment, the derivative is a prodrug.

The term “pharmaceutically acceptable salt(s)”, unless otherwiseindicated, includes salts of acidic or basic groups that may be presentin sialic acid or the sialic acid precursor of the present disclosure.

The terms “about” and “approximately” shall generally mean an acceptabledegree of error or variation for the quantity measured given the natureor precision of the measurements. Typical, exemplary degrees of error orvariation are within 20 percent (%), preferably within 10%, and morepreferably within 5% of a given value or range of values. For biologicalsystems, the term “about” refers to an acceptable standard deviation oferror, preferably not more than 2-fold of a give value. Numericalquantities given herein are approximate unless stated otherwise, meaningthat the term “about” or “approximately” can be inferred when notexpressly stated.

Methods of Treatment and Prevention

The present disclosure provides methods of treatment and/or preventionof nephrotic syndrome. The present disclosure further provides methodsof treatment and/or prevention of MCD. The present disclosureadditionally provides methods of alleviating one or more symptoms ofnephritic syndrome, such as, but not limited to, proteinuria,hypercholesterolemia, hypertriglyceridemia and edema. Still further, thepresent disclosure provides methods for reducing proteinuria. Furtherstill, the present disclosure provides methods for reducing edema. Thepresent disclosure also provides methods for reducinghypercholesterolemia and hypertriglyceridemia The present disclosurealso provides methods for the treatment and/or prevention of a diabeticcondition or a physiological condition associated therewith. The presentdisclosure additionally provides for pharmaceutical compositionscomprising sialic acid or one or more sialic acid precursors orcombinations of the foregoing.

In one embodiment, the teachings of the present disclosure provide forthe treatment and/or prevention of nephrotic syndrome in a subject inneed of such treatment or prevention. In one embodiment, the nephroticsyndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabeticnephropathy. In another embodiment, the nephrotic syndrome ischaracterized as MCD. Such method comprises the step of administeringsialic acid or a sialic acid precursor to the subject. Suchadministration restores normal sialylation of a polypeptide, such as,but not limited to Angptl4, involved in the etiology of nephroticsyndrome. Such administration thereby treats and/or prevents nephroticsyndrome in the subject. The sialic acid or sialic acid precursor may beadministered at a therapeutically effective amount. Furthermore, sialicacid or the sialic acid precursor may be administered alone, as a partof a pharmaceutical composition or in combination with a secondaryagent. Such method may further comprise identifying a subject in need ofsuch treatment and/or prevention.

In an alternate embodiment, the teachings of the present disclosureprovide for the treatment and/or prevention of MCD in a subject in needof such treatment or prevention. Such method comprises the step ofadministering sialic acid or a sialic acid precursor to the subject.Such administration restores normal sialylation of a polypeptide, suchas, but not limited to Angptl4, involved in the etiology of MCD. Suchadministration thereby treats and/or prevents MCD in the subject. Thesialic acid or sialic acid precursor may be administered at atherapeutically effective amount. Furthermore, sialic acid or the sialicacid precursor may be administered alone, as a part of a pharmaceuticalcomposition or in combination with a secondary agent. Such method mayfurther comprise identifying a subject in need of such treatment and/orprevention.

In further embodiment, the teachings of the present disclosure providefor methods of alleviating one or more symptoms of nephrotic syndrome,such as, but not limited to, proteinuria, hypercholesterolemia,hypertriglyceridemia and edema. In one embodiment, the nephroticsyndrome is characterized as MCD, FSGS, MN/MGN, MPGN or diabeticnephroapthy. In another embodiment, the nephrotic syndrome ischaracterized as MCD. Such method comprises the step of administeringsialic acid or a sialic acid precursor to the subject. Suchadministration restores normal sialylation of a polypeptide, such as,but not limited to Angptl4, involved in the etiology of nephroticsyndrome. Such administration thereby alleviates one or more symptoms ofnephrotic syndrome, such as, but not limited to, proteinuria and edema,nephrotic syndrome in the subject. The sialic acid or sialic acidprecursor may be administered at a therapeutically effective amount.Furthermore, sialic acid or the sialic acid precursor may beadministered alone, as a part of a pharmaceutical composition or incombination with a secondary agent. Such method may further compriseidentifying a subject in need of such treatment and/or prevention.

In still a further embodiment, the teachings of the present disclosureprovide methods for reducing proteinuria in a subject. In oneembodiment, the subject is suffering from nephrotic syndrome. In oneembodiment, the nephrotic syndrome is characterized as MCD, FSGS,MN/MGN, MPGN or diabetic nephroapthy. In another embodiment, thenephrotic syndrome is characterized as MCD. In another embodiment, thesubject is suffering from a disorder characterized by proteinuria, suchas, but not limited to, diabetic nephropathy, diabetes mellitus, lupusnephritis or primary glomular disease. In a specific embodiment, theproteinuria is caused, at least in part, by hyposialylation of apolypeptide, such as, but not limited to, Angptl4. Such method comprisesthe step of administering sialic acid or a sialic acid precursor to thesubject. Such administration restores normal sialylation of apolypeptide, such as, but not limited to Angptl4, involved in theetiology of proteinuria. Such administration thereby reduces proteinuriain the subject. The sialic acid or sialic acid precursor may beadministered at a therapeutically effective amount. Furthermore, sialicacid or the sialic acid precursor may be administered alone, as a partof a pharmaceutical composition or in combination with a secondaryagent. Such method may further comprise identifying a subject in need ofsuch reduction.

In yet a further embodiment, the teachings of the present disclosureprovide methods for reducing edema in a subject. In one embodiment, thesubject is suffering from nephrotic syndrome. In one embodiment, thenephrotic syndrome is characterized as MCD, FSGS, MN/MGN, MPGN ordiabetic nephropathy. In another embodiment, the nephrotic syndrome ischaracterized as MCD. In a specific embodiment, the edema is caused, atleast in part, by hyposialylation of a polypeptide, such as, but notlimited to, Angptl4. Such method comprises the step of administeringsialic acid or a sialic acid precursor or a combination of the foregoingto the subject. Such administration restores normal sialylation of apolypeptide, such as, but not limited to Angptl4, involved in theetiology of proteinuria. Such administration thereby reduces proteinuriain the subject. The sialic acid or sialic acid precursor may beadministered at a therapeutically effective amount. Furthermore, sialicacid or the sialic acid precursor may be administered alone, as a partof a pharmaceutical composition or in combination with a secondaryagent. Such method may further comprise identifying a subject in need ofsuch reduction.

In yet a further embodiment, the teachings of the present disclosureprovide methods for reducing hypercholesterolemia in a subject. In oneembodiment, the subject is suffering from nephrotic syndrome. In oneembodiment, the nephrotic syndrome is characterized as MCD, FSGS,MN/MGN, MPGN or diabetic nephropathy. In another embodiment, thenephrotic syndrome is characterized as MCD. In a specific embodiment,the hypercholesterolemia is caused, at least in part, by hyposialylationof a polypeptide, such as, but not limited to, Angptl4. Such methodcomprises the step of administering sialic acid or a sialic acidprecursor to the subject. Such administration restores normalsialylation of a polypeptide, such as, but not limited to Angptl4,involved in the etiology of hypercholesterolemia. Such administrationthereby reduces hypercholesterolemia in the subject. The sialic acid orsialic acid precursor may be administered at a therapeutically effectiveamount. Furthermore, sialic acid or the sialic acid precursor may beadministered alone, as a part of a pharmaceutical composition or incombination with a secondary agent. Such method may further compriseidentifying a subject in need of such reduction.

In yet a further embodiment, the teachings of the present disclosureprovide methods for reducing hypertriglyceridemia in a subject. In oneembodiment, the subject is suffering from nephrotic syndrome. In oneembodiment, the nephrotic syndrome is characterized as MCD, FSGS,MN/MGN, MPGN or diabetic nephropathy. In another embodiment, thenephrotic syndrome is characterized as MCD. In a specific embodiment,the hypertriglyceridemia is caused, at least in part, by hyposialylationof a polypeptide, such as, but not limited to, Angptl4. Such methodcomprises the step of administering sialic acid or a sialic acidprecursor to the subject. Such administration restores normalsialylation of a polypeptide, such as, but not limited, to Angptl4,involved in the etiology of hypertriglyceridemia. Such administrationthereby reduces hypertriglyceridemia in the subject. The sialic acid orsialic acid precursor may be administered at a therapeutically effectiveamount. Furthermore, sialic acid or the sialic acid precursor may beadministered alone, as a part of a pharmaceutical composition or incombination with a secondary agent. Such method may further compriseidentifying a subject in need of such reduction.

In yet a further embodiment, the teachings of the present disclosureprovide methods for treatment and/or prevention of a diabetic conditionin a subject or a physiological condition associated therewith. In oneembodiment, the diabetic condition is diabetic nephropathy, diabetesmellitus, lupus nephritis or primary glomular disease. In a specificembodiment, the diabetic condition is diabetic nephropathy. In oneembodiment, the physiological condition associated with the diabeticcondition is proteinuria. In a specific embodiment, the diabeticcondition is caused, at least in part, by hyposialylation of apolypeptide, such as, but not limited to, Angptl4. Such method comprisesthe step of administering sialic acid or a sialic acid precursor to thesubject. Such administration restores normal sialylation of apolypeptide, such as, but not limited to Angptl4, involved in theetiology of the diabetic condition. Such administration thereby treatsand/or prevents the diabetic condition in the subject. The sialic acidor sialic acid precursor may be administered at a therapeuticallyeffective amount. Furthermore, sialic acid or the sialic acid precursormay be administered alone, as a part of a pharmaceutical composition orin combination with a secondary agent. Such method may further compriseidentifying a subject in need of such treatment and/or prevention.

Methods of Diagnosis

The present disclosure also provides methods for determining the statusof a subject with respect to nephrotic syndrome or a diabetic condition.In one embodiment, the nephrotic syndrome is characterized as MCD, FSGS,MN/MGN, MPGN or diabetic nephropathy. In another embodiment, thenephrotic syndrome is characterized as MCD. In one embodiment, thediabetic condition is diabetic nephropathy, diabetes mellitus, lupusnephritis or primary glomular disease. In a specific embodiment, thediabetic condition is diabetic nephropathy.

In one embodiment, such methods determine in the subject the level of apolypeptide associated with nephrotic syndrome or a diabetic condition,such as, but not limited to, Angptl4, the level of sialylation of apolypeptide associated with nephrotic syndrome or a diabetic condition,such as, but not limited to, Angptl4, or a combination of the foregoing.In a particular embodiment, the level of the high pI form of thepolypeptide is determined. This form has been shown to be hyposialylatedand indicative of nephrotic syndrome or a diabetic condition. The amountand/or level of sialylation of the polypeptide as determined from thesubject may be compared to corresponding amounts and levels from asubject that is diagnosed as not suffering from nephrotic syndrome or adiabetic condition (control subject). Such amounts and levels may alsobe compared to a reference standard. A decrease in the level ofsialylation as compared to the control subject or reference standardindicates the subject is suffering from or at risk for, nephroticsyndrome or a diabetic condition; as discussed above, the level ofsialylation may be determined with respect to the high pI form of thepolypeptide (which is hyposialylated).

The present disclosure further provides methods for determining theefficacy of a treatment for nephrotic syndrome or a diabetic conditionin a subject undergoing treatment for nephrotic syndrome or a diabeticcondition. In one embodiment, the nephrotic syndrome is characterized asMCD, FSGS, MN/MGN, MPGN or diabetic nephropathy. In another embodiment,the nephrotic syndrome is characterized as MCD. In one embodiment, thediabetic condition is diabetic nephropathy, diabetes mellitus, lupusnephritis or primary glomular disease. In a specific embodiment, thediabetic condition is diabetic nephropathy.

In one embodiment, such methods determine in the subject the level of apolypeptide associated with nephrotic syndrome or a diabetic condition,such as, but not limited to, Angptl4, the level of sialylation of apolypeptide associated with nephrotic syndrome or a diabetic condition,such as, but not limited to, Angptl4, or a combination of the foregoing.In a particular embodiment, the level of the high pI form of thepolypeptide is determined. This form has been shown to be hyposialylatedand indicative of nephrotic syndrome or a diabetic condition. The amountand/or level of sialylation of the polypeptide as determined from thesubject during treatment may be compared to corresponding amounts and/orlevels from the subject prior to initiating treatment. An increase inthe level of sialylation in the subject undergoing treatment as comparedto the level of sialylation determined prior to initiating treatmentindicates the treatment is having the desired effect; as discussedabove, the level of sialylation may be determined with respect to thehigh pI form of the polypeptide (which is hyposialylated). Furthermore,the amount and/or level of the polypeptide as determined from thesubject during treatment may be compared to corresponding amounts and/orlevels a subject that is diagnosed as not suffering from nephroticsyndrome or a diabetic condition (control subject). Such amounts andlevels may also be compared to a reference standard. A level ofsialylation obtained from the subject during treatment that is equal toor approaching the level of sialylation from the control subject orreference indicates the treatment is having the desired effect; asdiscussed above, the level of sialylation may be determined with respectto the high pI form of the polypeptide (which is hyposialylated).

Any method known in the art for determining protein levels and levels ofsialylation may be used in such methods, including, but not limited to,any methods described herein.

FIG. 5C shows the utility of this approach. 200 μg human plasma frompatients (n=4 patients/group) with various conditions were analyzed by2D gel electrophoresis and Western blots were prepared usinganti-Angptl4 antibodies. This figure demonstrates that only patientswith MCD relapse had circulating hyposialylated form of Angptl4polypeptide (this 55-70 kDa pI 8-8.5 form of the Angptl4 polypeptide isindicated by the oval); this polypeptide was absent in patients with MCDremission. Increased circulating neutral pI monomers and oligomers werenoted in MCD patients in relapse (arrow), and monomers only in MN(arrow).

Any of the above methods of treatment may be used in conjunction withthe methods of administering a polypeptide described in U.S. provisionalApplication Nos. 61/351,866 and 61/483,854, which are herebyincorporated by reference in their entirety.

Methods of Screening

The present disclosure also relates to a method for identifying acompound effective for treating or preventing nephrotic syndrome, adiabetic condition or a condition associated therewith, such as, but notlimited to, proteinuria, hypercholesterolemia, hypertriglyceridemia oredema. In one embodiment, the nephrotic syndrome is characterized asMCD, FSGS, MN or MGN. In another embodiment, the nephrotic syndrome ischaracterized as MCD. In one embodiment, the diabetic condition isdiabetic nephropathy, diabetes mellitus, lupus nephritis or primaryglomular disease. In a specific embodiment, the diabetic condition isdiabetic nephropathy. Such compounds may be useful as active ingredientsincluded in pharmaceutical compositions or for administration alone. Inone embodiment, the methods include determining the level of sialylationof a polypeptide involved in the etiology of nephrotic syndrome, suchas, but not limited to, Angptl4.

In general, such screening methods comprises the steps of providing anassay system (as described in more detail below) that expresses apolypeptide involved in the etiology of nephrotic syndrome or a diabeticcondition, such as, but not limited to, Angptl4, introducing into theassay system a test compound to be tested and determining whether theeffect of the test compound on the level of sialylation of thepolypeptide. The methods involve the identification of candidate or testcompounds or agents (polypeptides, functional nucleic acids,carbohydrates, antibodies, small molecules or other molecules) whicheffect the level of sialylation of the polypeptide. Such compounds maythen be further tested in appropriate systems (such as, but not limitedto, the animal models systems described herein) to determine theactivity of the identified compounds.

Candidate compounds are identified using a variety of assays, such as,but not limited to, assays that employ cells which express a polypeptideinvolved in the etiology of nephrotic syndrome or a diabetic condition,such as, but not limited to, Angptl4 or in assays with isolatedpolypeptides. The various assays can employ a variety of variants ofsuch polypeptides (e.g., full-length, a biologically active fragment, ora fusion protein which includes all or a portion of the desiredpolypeptide). Moreover, such polypeptides can be derived from anysuitable mammalian species (e.g., human, rat or murine); in a specificembodiment, the polypeptide is derived from a human.

Where the assay involves the use of a whole cell, the cell may eithernaturally express a polypeptide involved in the etiology of nephroticsyndrome or a diabetic condition, such as, but not limited to, Angptl4,or may be modified to express the same. In the latter case, cells can bemodified to express a desired polypeptide through conventional molecularbiology techniques, such as by infecting the cell with a viruscomprising such polypeptide. The cell can also be a prokaryotic or aneukaryotic cell that has been transfected with a nucleotide sequenceencoding such polypeptide. In the foregoing, full length polypeptides,fragments or fusion proteins containing at least a part of suchpolypeptide may be used. Exemplary assay systems are described in thecurrent specification.

The various screening assays may be combined with an in vivo assayentailing measuring the effect of the test compound on the symptoms thedisease states and conditions discussed herein. In such an embodiment,the compounds may be evaluated to determine if they impact a parameterassociated with nephrotic syndrome or a diabetic condition or acondition related thereto, such as, but not limited to, proteinuriahypercholesterolemia, hypertriglyceridemia or edema. Such parametersinclude, but are not limited to, determining 1) the level of sialylationof a polypeptide involved in the etiology of hypercholesterolemia,reduces hypertriglyceridemia or related conditions, such as, but notlimited to Angptl4 and 2) the amount of high pI forms a polypeptideinvolved in the etiology of hypercholesterolemia, reduceshypertriglyceridemia or related conditions, such as, but not limited toAngptl4 (these forms are hyposialylated); 3) determining the level ofprotein excretion, either total or with regard to specific components;and 4) determining the impact of the test compound on kidney morphology,such as, but not limited, the podocyte.

In one embodiment, such a screening assay can be performed, for example,by determining the level of sialylation of a polypeptide involved in theetiology of hypercholesterolemia, reduces hypertriglyceridemia, such as,but not limited to, Angptl4 and detecting a difference in the level ofsialylation of such polypeptide in the presence of as compared to theabsence of a test compound. In a particular embodiment, the high pIforms of such polypeptide are specifically examined (this form ishyposialylated). Such screening assay may be in vitro, in vivo or exvivo and may be cell culture based (either with whole cells or lysates)or may be based on an animal model. Any assay of the present disclosuremay be used in the foregoing method.

Suitable test compounds for use in the screening methods can be obtainedfrom any suitable source, such as conventional compound libraries. Thetest compounds can also be obtained using any of the numerous approachesin combinatorial library methods known in the art, including: biologicallibraries, spatially addressable parallel solid phase or solution phaselibraries, synthetic library methods requiring deconvolution, the“one-bead one-compound” library method and synthetic library methodsusing affinity chromatography selection. The biological library approachis limited to peptide libraries, while the other four approaches areapplicable to peptide, non-peptide oligomer or small molecule librariesof compounds. Examples of methods for the synthesis of molecularlibraries can be found in the art. Libraries of compounds may bepresented in solution or on beads, bacteria, spores, plasmids or phage.

In the foregoing methods, the high pI forms of Angptl4 refer to apolypeptide migrating at 55-70 kDa and having a pI of 8-8.5.

The present disclosure also provides kits for carrying out any method ofthe present disclosure, which can contain any of the compounds and/orcompositions disclosed herein or otherwise useful for practicing amethod of the disclosure.

Sialic Acid Precursors

The present disclosure provides for various uses of sialic acid(N-acetylneuraminic acid) or a sialic acid precursor.

In one embodiment, the sialic acid precursor is N-acetylmannosamine(ManNAc; also referred to as 2-Acetamido-2-deoxy-D-mannose orN-acetyl-D-mannosamine). The structure of ManNAc is shown in formula Ibelow.

In an alternate embodiment, the sialic acid precursor is defined by theformula Ia below.

Wherein:

-   A is CH₂ or NH;-   B, C, D and E are each independently selected from the group    consisting of: H, OH, X, O—CO—X or O—X, wherein X is a substituted    or unsubstituted alkyl or alkenyl, X (when present) being selected    independently for each group B, C, D and E;-   G is H, OH, Y or O—Y, wherein Y is a substituted or unsubstituted    alkyl or alkenyl.

It is understood that the compounds of Formula I and Ia can also berepresented in the chair configuration as well.

In one embodiment, X and/or Y are independently a C1 to C5 alkyl. In aparticular embodiment, X and/or Y are propyl; in a further embodiment, Xand/or Y are butyl.

In one embodiment, at least one of B, C, D, E or G is not H or OH. In afurther embodiment, at least two of B, C, D, E or G is not H or OH. Instill a further embodiment, at least three of B, C, D, E or G is not Hor OH. In yet another embodiment, none of B, C, D, E or G is not H orOH.

In a particular embodiment, G is CH₃, A is CH₂, and at least one of B,C, D or E is not H or OH. In a further particular embodiment, G is CH₃,A is CH₂, and at least two of B, C, D or E are not H or OH. In still afurther particular embodiment, G is CH₃, A is CH₂, and at least three ofB, C, D or E are not II or OII. In yet a further particular embodiment,G is CH₃, A is CH₂, and all of B, C, D or E are not H or OH. In theforegoing, in one embodiment, B, C and D may each independently be CO—X.

It is noted that where G is CH₃, A is CH₂, and B, C, D and E are eachOH, the compound is ManNAc (Formula I).

Representative derivatives falling under the formula I include, but arenot limited to, Bu4ManNAc, 3,4,6—O—Bu3ManNAc or 1,3,4—O—Bu3ManNAc (suchcompounds are described in Aich, et al, Glycoconj J. DOI10.1007/s10719-010-9292-3 accepted Apr. 14, 2010).

Additional sialic acid precursors include N-levulinoyl sialic acid(SiaLev) and N-levulinoylmannosamine (ManLev) (Charter et al.Glycobiology. 2000 October; 10 (10):1049-56).

All enantiomeric fauns of the foregoing compounds are included in theabove descriptions.

Compositions

Useful compositions of the present disclosure comprise one or morecompounds useful in the treatment and prevention methods of the presentdisclosure, such as, but not limited to, sialic acid, sialic acidprecursors, those compounds identified in the present disclosure oridentified by a screening method of the present disclosure. In oneembodiment, such compounds increase the sialylation of a polypeptide,such as, but not limited to, Angptl4. In a particular embodiment, thecompounds are sialic acid precursors. Exemplary sialic acid precursorsinclude, but are not limited to, ManNAc and ManNAc derivatives.

The compositions disclosed may comprise one or more of such compounds,in combination with a pharmaceutically acceptable carrier. Examples ofsuch carriers and methods of formulation may be found in Remington: TheScience and Practice of Pharmacy (20^(th) Ed., Lippincott, Williams &Wilkins, Daniel Limmer, editor). To form a pharmaceutically acceptablecomposition suitable for administration, such compositions will containan therapeutically effective amount of compound.

The pharmaceutical compositions of the disclosure may be used in thetreatment and prevention methods of the present disclosure. Suchcompositions are administered to a subject in amounts sufficient todeliver a therapeutically effective amount of the compound(s) so as tobe effective in the treatment and prevention methods disclosed herein.The therapeutically effective amount may vary according to a variety offactors such as, but not limited to, the subject's condition, weight,sex and age. Other factors include the mode and site of administration.The compositions of the present disclosure may be administered only onetime to the subject or more than one time to the subject. Furthermore,when the compositions are administered to the subject more than once, avariety of regimens may be used, such as, but not limited to, one perday, once per week or once per month. The compositions may also beadministered to the subject more than one time per day. Thetherapeutically effective amount and appropriate dosing regimens may beidentified by routine testing in order to obtain optimal activity, whileminimizing any potential side effects. In addition, co-administration orsequential administration of other agents may be desirable.

The compositions of the present disclosure may be administered in avariety of dosage forms and regimens as discussed herein. Exemplarydoses include, but are not limited to, of at least about 0.1 mg/kg toabout 750 mg/kg, of at least about 1 mg/kg to about 500 mg/kg, at leastabout 1 mg/kg to about 200 mg/kg or at least about 1 mg/kg to about 100mg/kg of body weight. Daily doses of a sailic acid precursor may rangefrom about 0.1 g/day to about 75 g/day, from about 0.5 g/day to about 50g/day, from about 1 g/day to about 10 g/day, from about 0.1 g/day toabout 5 g/day, from about 0.1 g/day to about 3 g/day, and from about 0.1g/day to about 1 g/day.

The pharmaceutical compositions may be provided to the subject in anymethod known in the art. Exemplary routes of administration include, butare not limited to, oral, subcutaneous, rectal, parenteral,subcutaneous, intramuscular, intraperitoneal, intravenous, topical,epicutaneous, intraosseous, intramuscular, dermal, transdermal,intrathoracic, intrapulmonary, intranasal or pulmonary routes

The compositions of the present disclosure may further comprise agentswhich improve the solubility, half-life, absorption, etc. of thecompound(s). Furthermore, the compositions of the present disclosure mayfurther comprise agents that attenuate undesirable side effects and/oror decrease the toxicity of the compounds(s). Examples of such agentsare described in a variety of texts, such a, but not limited to,Remington: The Science and Practice of Pharmacy (20^(th) Ed.,Lippincott, Williams & Wilkins, Daniel Limmer, editor).

The compositions of the present disclosure can be administered in a widevariety of dosage forms for administration. For example, thecompositions can be administered in forms, such as, but not limited to,tablets, capsules, sachets, lozenges, troches, pills, powders, granules,elixirs, tinctures, solutions, suspensions, elixirs, syrups, ointments,creams, pastes, emulsions, or solutions for intravenous administrationor injection. Other dosage forms include administration transdermally,via patch mechanism or ointment. Any of the foregoing may be modified toprovide for timed release and/or sustained release formulations.

In the present disclosure, the pharmaceutical compositions may furthercomprise a pharmaceutically acceptable carriers include, but are notlimited to, vehicles, adjuvants, surfactants, suspending agents,emulsifying agents, inert fillers, diluents, excipients, wetting agents,binders, lubricants, buffering agents, disintegrating agents andcarriers, as well as accessory agents, such as, but not limited to,coloring agents and flavoring agents (collectively referred to herein asa carrier). Typically, the pharmaceutically acceptable carrier ischemically inert to the active compounds and has no detrimental sideeffects or toxicity under the conditions of use. The pharmaceuticallyacceptable carriers can include polymers and polymer matrices. Thenature of the pharmaceutically acceptable carrier may differ dependingon the particular dosage form employed and other characteristics of thecomposition.

For instance, for oral administration in solid form, such as but notlimited to, tablets, capsules, sachets, lozenges, troches, pills,powders, or granules, the compound(s) may be combined with an oral,non-toxic pharmaceutically acceptable inert carrier, such as, but notlimited to, inert fillers, suitable binders, lubricants, disintegratingagents and accessory agents. Suitable binders include, withoutlimitation, starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,polyethylene glycol, waxes and the like. Lubricants used in these dosageforms include, without limitation, sodium oleate, sodium stearate,magnesium stearate, sodium benzoate, sodium acetate, and the like.Disintegrators include, without limitation, starch, methyl cellulose,agar, bentonite, xanthum gum and the like. Tablet forms can include oneor more of the following: lactose, sucrose, mannitol, corn starch,potato starch, alginic acid, microcrystalline cellulose, acacia,gelatin, guar gum, colloidal silicon dioxide, croscarmellose sodium,talc, magnesium stearate, calcium stearate, zinc stearate, stearic acidas well as the other carriers described herein. Lozenge forms cancomprise the active ingredient in a flavor, usually sucrose and acaciaor tragacanth, as well as pastilles comprising the active ingredient inan inert base, such as gelatin and glycerin, or sucrose and acadia,emulsions, and gels containing, in addition to the active ingredient,such carriers as are known in the art.

For oral liquid forms, such as but not limited to, tinctures, solutions,suspensions, elixirs, syrups, the nucleic acid molecules of the presentdisclosure can be dissolved in diluents, such as water, saline, oralcohols. Furthermore, the oral liquid forms may comprise suitablyflavored suspending or dispersing agents such as the synthetic andnatural gums, for example, tragacanth, acacia, methylcellulose and thelike. Moreover, when desired or necessary, suitable and coloring agentsor other accessory agents can also be incorporated into the mixture.Other dispersing agents that may be employed include glycerin and thelike.

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the patient, and aqueous andnon-aqueous sterile suspensions that can include suspending agents,solubilizers, thickening agents, stabilizers, and preservatives. Thecompound(s) may be administered in a physiologically acceptable diluent,such as a sterile liquid or mixture of liquids, including water, saline,aqueous dextrose and related sugar solutions, an alcohol, such asethanol, isopropanol, or hexadecyl alcohol, glycols, such as propyleneglycol or polyethylene glycol such as poly(ethyleneglycol) 400, glycerolketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, an oil, afatty acid, a fatty acid ester or glyceride, or an acetylated fatty acidglyceride with or without the addition of a pharmaceutically acceptablesurfactant, such as, but not limited to, a soap, an oil or a detergent,suspending agent, such as, but not limited to, pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations, include petroleum,animal, vegetable, or synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includepolyethylene sorbitan fatty acid esters, such as sorbitan monooleate andthe high molecular weight adducts of ethylene oxide with a hydrophobicbase, formed by the condensation of propylene oxide with propyleneglycol, oleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters. Suitablesoaps for use in parenteral formulations include fatty alkali metal,ammonium, and triethanolamine salts, and suitable detergents include (a)cationic detergents such as, for example, dimethyldialkylammoniumhalides, and alkylpyridinium halides, (b) anionic detergents such as,for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether,and monoglyceride sulfates, and sulfosuccinates, (c) nonionic detergentssuch as, for example, fatty amine oxides, fatty acid alkanolamides, andpolyoxyethylene polypropylene copolymers, (d) amphoteric detergents suchas, for example, alkylbeta-aminopropionates, and 2-alkylimidazolinequaternary ammonium salts, and (e) mixtures thereof.

Suitable preservatives and buffers can be used in such formulations. Inorder to minimize or eliminate irritation at the site of injection, suchcompositions may contain one or more nonionic surfactants having ahydrophile-lipophile balance (HLB) of from about 12 to about 17. Thequantity of surfactant in such formulations ranges from about 5% toabout 15% by weight.

Topical dosage forms, such as, but not limited to, ointments, creams,pastes, emulsions, containing the nucleic acid molecule of the presentdisclosure, can be admixed with a variety of carrier materials wellknown in the art, such as, e.g., alcohols, aloe vera gel, allantoin,glycerine, vitamin A and E oils, mineral oil, PPG2 myristyl propionate,and the like, to form alcoholic solutions, topical cleansers, cleansingcreams, skin gels, skin lotions, and shampoos in cream or gelformulations. Inclusion of a skin exfoliant or dermal abrasivepreparation may also be used. Such topical preparations may be appliedto a patch, bandage or dressing for transdermal delivery or may beapplied to a bandage or dressing for delivery directly to the site of awound or cutaneous injury.

The compound(s) of the present disclosure can also be administered inthe form of liposome delivery systems, such as small unilamellarvesicles, large unilamellar vesicles and multilamellar vesicles.Liposomes can be formed from a variety of phospholipids, such ascholesterol, stearylamine or phosphatidylcholines. Such liposomes mayalso contain monoclonal antibodies to direct delivery of the liposome toa particular cell type or group of cell types.

The compound(s) of the present disclosure may also be coupled withsoluble polymers as targetable drug carriers. Such polymers can include,but are not limited to, polyvinyl-pyrrolidone, pyran copolymer,polyhydroxypropylmethacryl-amidephenol,polyhydroxyethylaspartamidephenol, or polyethyl-eneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydro-pyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels.

Furthermore, the sialic acid or sialic acid precursors disclosed hereincan be administered as a food supplement or incorporated into food ordrink items.

Results

In the following results, the methods used were those methods specifiedin the Methods section of the present disclosure and the referencescited therein.

Upregulation of Podocyte Angptl4 Expression in Experimental and HumanGlomerular Disease

Angptl4 mRNA expression is upregulated (70-fold) in rat glomeruli at thepeak of complement- and leukocyte-independent heterologous phaseproteinuria 24 hours after injection of NTS (FIG. 1A and 1B). Injectionof NTS into Angptl4 −/− mice caused significant reduction in proteinuria(FIG. 1C) and foot process effacement (FIG. 1D), suggesting a key rolefor Angptl4 in glomerular disease. Normal rat glomeruli express Angptl4in a capillary loop pattern that co-localized with podocyte proteinCD2AP (FIG. 1E). Further studies revealed early (Day 3, before the onsetof proteinuria) and progressive upregulation of Angptl4 mRNA expressionin young rats following intravenous injection of a single dose ofpuromycin aminonucleoside (PAN model) (FIG. 1F), and in situhybridization confirmed upregulation in a peripheral capillary looppattern (data not shown) without concomitant change in the proximaltubular signal. In passive Heymann nephritis, a smaller increase inAngptl4 expression was noted, starting after the onset of proteinuria(FIG. 1F). By contrast, Angptl4 mRNA expression did not change inanti-Thy1.1 nephritis, or in collapsing focal and segmentalglomerulosclerosis (FIG. 1F) induced in rats by injection of sera frompatients with this disease (18). Angptl4 protein expression increaseddramatically in podocytes (FIG. 1G) following induction of PAN, withsubstantial additional overlap with the glomerular basement membrane(GBM), which was confirmed by immunogold electron microscopy (EM) (FIG.1H). Biopsies from patients with glucocorticoid sensitive MCD and ageand sex-matched controls (FIG. 1I) revealed a faint podocyte pattern incontrol kidney biopsies, and increased expression in the podocyte withadditional GBM overlap and trace spotty overlap with the endothelium atthe margins.

Podocyte Specific Overexpression of Angptl4 Results in SelectiveProteinuria and Podocyte Foot Process Effacement

A transgenic rat models for podocyte specific Angptl4 overexpression wasdeveloped and is shown in FIG. 2A (NPHS2-Angptl4 TG). Analysis of mRNAexpression in organs that normally express Angptl4 confirmed specificityof expression (FIG. 2B). Histological assessment of 3 month oldheterozygous male NPHS2-Angptl4 TG rats revealed normal appearingglomeruli with prominent podocytes (FIG. 2C) on light microscopy andincreased podocyte Angptl4 expression by confocal imaging (FIG. 2D).Electron microscopy of 5 month old homozygous and most heterozygous TGrats (FIG. 2E) revealed diffuse foot process effacement. Immunogold EMof homozygous TG rats revealed a correlation between accumulation ofAngptl4 in the GBM and progressive development of foot processeffacement between age one to five months (FIG. 2F).

Both founder lines of NPHS2-Angptl4 TG rats developed significantalbuminuria. Female homozygous and male heterozygous rats developedalbuminuria as early as age 1 month (FIGS. 3A-3C). Homozygous femalesdeveloped up to 100-fold, heterozygous males up to 20-fold andhomozygous males over 500-fold increase in albuminuria over time.Heterozygous females were not albuminuric. Over 90% of the urinaryprotein comprises of intact albumin (FIG. 3D), thereby making these ratsthe first model for selective proteinuria. Immunogold EM using anti-V5antibody to specifically detect transgene expressed protein revealedgold particles in the podocyte and GBM in NPHS2-Angptl4 TG rats (FIG.3E). In keeping with the pro-proteinuric effects of podocyte secretedAngptl4, NPHS2-Angptl4 TG rats develop more proteinuria (FIG. 3F) thanwild type littermates after induction of PAN. Blood pressure wassignificantly lower in proteinuric heterozygous NPHS2-Angptl4 ratscompared to wild type controls (data not shown). As also previouslypublished (20), proteinuria in PAN is partially glucocorticoid sensitiveon day 6 (FIG. 3G), and some of this glucocorticoid sensitivity isrelated to Angptl4 (FIG. 3H).

Characterization of Podocyte Secreted and Recombinant Angptl4

Glomerular protein Western blot typically underestimates Angptl4production, since the protein is rapidly secreted. Angptl4 was analyzedby 2D electrophoresis and Western blotting (FIG. 4A) in glomeruli fromcontrol (upper panel), a PAN model at day 6 (middle panel) and a PANmodel at day 6 with glucocorticoid coadministration (lower panel). On 2Dgel electrophoresis, most Angptl4 in normal glomeruli migrates asglycosylated low order oligomers at neutral pI (pink arrow; 3), thoughless prominent spots for both glycosylated intact 70 KDa and cleaved(red arrow; 1) and non glycosylated 45 KDa monomeric forms (yellowarrow; 2) were also noted (FIGS. 4A and 4B). In PAN, both neutral pIoligomers, that are reactive with sialic acid binding lectin Maackiaamurensis (MAA), and high pI oligomers, that are not MAA reactive, wereincreased (pink arrow, 3 and orange arrow, 4) (FIGS. 4A and 4B). Thisincrease was blunted in PAN treated with glucocorticoids, thoughdisproportionately higher amounts of high pI oligomers were noted.Densitometric analysis of the Western blots is shown in FIG. 4B.

FIG. 5A shows HEK293 cells stably transfected with a pcDNA-ratAngptl4-V5-His expression construct line showed 55,000 fold upregulationof Angptl4 mRNA expression compared to a control empty vector cell line.*** P<0.001

FIG. 5B shows Angptl4-HEK293 cells secreted mostly intact protein intothe supernatant underserum free conditions (as demonstrated by 2D gelelectrophoresis and Western blot with pre-immune serum and anti-Angptl4antibodies).

Sialylation of Angptl4 was studied in vitro (FIG. 4C) using theAngptl4-HEK293 stable cell line. HEK293 cells normally express no orlittle Angptl4 polypeptide; after transfection with the pcDNA-ratAngptl4-V5-His expression construct, this cell line showed a 55,000 foldupregulation of Angptl4 mRNA expression compared to a control emptyvector cell line (FIG. 5A). Furthermore, the Angptl4 polypeptideexpressed by the HEK293-Angptl4 cell line is normally secreted by thiscell line is only trace MAA reactive at the low pI end (FIG. 4C upperpanel and upper middle panel, green arrow 1 and FIG. 5B), and istherefore mostly hyposialylated. Incubation of the cell line with sialicacid precursor N-Acetyl-D-mannosamine (ManNAc; FIG. 4C, lower middlepanel and lower panel) resulted in a shift of the protein towardsneutral pI, increased reactivity to MAA (blue arrow, 2), and therefore,increased sialylation, showing that sialylation plays a key role in thedifferential electrophoretic migration of Angptl4.

Since an increase in hyposialylated Angptl4 was noted in PAN, the roleof this lack of sialylation in the pathogenesis of proteinuria wasstudied. FIG. 6A shows representative tracing of albuminuria from pilotstudies with heavily albuminuric rats receiving increasing doses ofManNAc. The study revealed that ManNAc reduced albuminuria in theseanimals. However, a large dose requirement to reduce albuminuria inthese rats was required. In order to conduct a more efficient andindicative study, rats with 15-25 fold higher albuminuria than wild typerats were used for further analysis.

NPHS2-Angptl4 TG rats that received ManNAc (1 mg/ml) in tap water (FIG.4D) had over 40% reduction in albuminuria over a 12 day period comparedto baseline, whereas albuminuria in control NPHS2-Angptl4 TG ratsreceiving tap water increased 2.5 fold over the duration of the study.FIG. 6B shows individual tracings from the study group of NPHS2-Angptl4TG rats treated with ManNAc (1 mg/ml and which went through the completestudy. Two urine collections 12 days apart were conducted prior to thestart of the study to ensure that these rats developed increasingalbuminuria with time. The study revealed that ManNAc (1 mg/ml) reducedalbuminuria in these animals.

Densitometry analysis of Western blots for glomerular Angptl4 from ratseuthanized on Day 12 (FIGS. 6C and 6D) showed an increase in the neutralp1 Angptl4 fraction from 48.3+5% in control rats to 72.9+1.4% in ManNActreated rats (FIG. 6D). The neutral pI fraction was also reactive withsialic acid binding lectin Sambucus nigra (SNA I), conforming increasedsialylation of Angptl4 in ManNAc treated rats (FIG. 6C).

FIG. 7 shows improvements in nephrotic syndrome parameters in ManNActreated rats with PAN, a model of MCD. Rats (n=5 rats/group) received asingle intravenous injection of puromycin aminonucleoside (10 mg/100gm), and were treated with ManNAC 80 mg/Kg body weight in tap waterstarting on Day 4, which coincides with the onset of proteinuria. Datafrom Day 6 is shown. Significant reduction in proteinuria (FIG. 7A),improvement in plasma albumin levels (FIG. 7B), reduction in cholesterol(FIG. 7C) and reduction in triglycerides (FIG. 7D) were noted in ManNActreated rats when compared with control PAN rats. These data areconsistent with the results described above.

Proteinuria, including albuminuria, has been noted in other diseases asdiscussed herein, including diabetes mellitus. The role of Angptl4 indiabetes mellitus was also analyzed using the db/db mouse. The db/db wasidentified initially in 1966 in Jackson Labs as an obese mouse that washyperphagic soon on weaning. The diabetic gene (db) is transmitted as anautosomal recessive trait. The db gene encodes for a G-to-T pointmutation of the leptin receptor, leading to abnormal splicing anddefective signaling of the adipocyte-derived hormone leptin. Lack ofleptin signaling in the hypothalamus will lead to persistent hyperphagiaand obesity with consequently high leptin and insulin levels. Severalstudies established that albumin excretion rates are higher by 8- to62-fold in db/db mice beginning at the age of 8 wk. The range ofalbuminuria is between 68 and 303 μg/24 h in the db/db male mouse,whereas it is between 4 and 21 μg/24 h in the age-matched heterozygouslittermate.

FIGS. 8A-C show the effect of ManNAc therapy on proteinuria in diabeticanimals FIG. 8A shows 2D gel electrophoresis and Western blot analysisof glomeruli from diabetic db/db and control db/m mice. This analysisshowed increased expression of Angptl4 in diabetic db/db mouseglomeruli, including the high pI hyposialylated forms that are involvedin the pathogenesis of proteinuria. These high pI hyposialylated formsof Angptl4 are sensitive to sialic acid precursor therapy, includingManNAc and ManNAc derivatives.

FIG. 8B shows 2D gel electrophoresis and Western blot analysis ofglomeruli from Zucker diabetic fatty rats. This analysis showedincreased expression of Angptl4 in Zucker diabetic fatty rat glomeruli,including the high pI hyposialylated forms that are involved in thepathogenesis of proteinuria. These high pI hyposialylated forms ofAngptl4 are sensitive to sialic acid precursor therapy, including ManNAcand ManNAc derivatives. FIG. 8C shows treatment with a sialic acidprecursor reduced proteinuria in this model. In FIG. 8C, 13 week oldmale Zucker diabetic fatty rats were treated with ManNAc in tap water orwith plain tap water (n=4 rats/group). Significant reduction inproteinuria was noted in the ManNAc treated group for up to 20 days at70 mg/Kg and at an increased dose of 140 mg/Kg.

These results show that administration of sialic acid precursors treatsdiabetes mellitus through restoration of the normal sialyation ofAngptl4. As shown in FIG. 8C, administration of sialic acid precursorsreduces proteinuria observed in diabetes mellitus through restoration ofnormal sialylation of the Angptl4 polypeptide as described herein.

The present disclosure shows a key role of podocyte secreted Angptl4 inthe pathogenesis of nephrotic syndrome, exemplified using a model of MCDand the role of Angptl4 in conditions related to nephrotic syndrome,such as, but not limited to, proteinuria. The present disclosures showsincreased podocyte expression of Angptl4 in human kidney biopsies,selective proteinuria with over 500-fold increase in albuminuria inNPHS2-Angptl4 TG rats, glucocorticoid sensitivity of the Angptl4 gene,and light and electron microscopic findings consistent with human MCD.The role of Angptl4 in the development of proteinuria and foot processeffacement was further confirmed by the significant reduction inproteinuria and foot process effacement in Angptl4 −/− mice injectedwith NTS. The absence of a reliable model of MCD in mice necessitatedthe use of NTS.

The secretion of high pI Angptl4 by the podocyte in MCD is likely tofacilitate the tethering of Angptl4 to the GBM, and prior studies haveshown the binding of Angptl4 to heparan sulfate proteoglycans (21). Thiscorrelates well with reduced GBM charge, a hallmark of MCD, in rodentswith TG expression of Angptl4 from the podocyte. It is likely thatcharge facilitates the transit of high pI Angptl4 across the GBM againstthe direction of fluid flow. Changes in sialylation explain the bulk ofthe variations in pI and the biological role of Angptl4 in proteinuria,since the active oligomeric forms of Angptl4 secreted from podocyteswere not threonine phosphorylated, and increasing the sialylation ofAngptl4 reduced proteinuria. There are two predicted O- andN-glycosylation sites each in rat and human Angptl4 where sialic acidresidues could be incorporated. Treatment with sialic acid precursorsconstitute a new potential therapeutic tool to reduce proteinuria innephrotic syndrome, such as, but not limited to, MCD, and perhaps commonmultisystem disorders like diabetes mellitus, in which Angptl4 plays arole.

Additional, as yet undefined, podocyte or endothelial cell secretedfactors probably work synergistically with Angptl4 in the pathogenesisof MCD. In NPHS2-Angptl4 TG rats, podocyte secreted Angptl4 remainstethered to the glomerular capillary loop or escapes into the urinaryspace, where it is taken up by the proximal tubule. After induction oflow dose PAN in these rats, which provides these additionalpermeabilizing factors, podocyte secreted Angptl4 escapes into thecirculation. The lack of additional permeabilizing factors inNPHS2-Angptl4 TG rats is also likely to also explain the more gradualonset of proteinuria than in PAN or patients with MCD.

The foregoing shows that administration of a sialic acid precursorrestores normal sialylation of Angplt4 polypeptide and reducesphysiological abnormalities associated nephrotic syndrome (including,but not limited to, MCD, FSGS, MN/MGN, MPGN or diabetic nephropathy),diabetes mellitus, lupus nephritis or primary glomular disease. As aresult, the present disclosure provides a treatment for such conditions.

Methods Cloning of Full Length Rat Angptl4, and Generation of AntibodyAgainst Full Length Recombinant Angptl4

The full length rat Angptl4 open reading frame of 1218 bp from ourprevious experiments (7), excluding the stop codon, was cloned intopcDNA3.1/V5-HisB for eukaryotic expression, and into pET28a forprokaryotic expression. The E. Coli expressed purified full lengthprotein was used to generate a polyclonal antibody in rabbits(Proteintech group, Inc. Chicago Ill. USA) that was tested by ELISA andWestern blot. Antibody reactive bands were excised from GelCode bluestained gels, trypsin digested and presence of Angptl4 peptide sequencesconfirmed by MALDI-TOF/TOF. Part of the antiserum was affinity purifiedto the antigen. Unless otherwise specified, all studies described usedthis antibody. An additional polyclonal antibody against the N-terminalpart of rat Angptl4 (amino acids 7-86 excluding signal peptide) wassimilarly raised in rabbits.

Induction of Proteinuria in Animal Models of Human Glomerular Disease

All animal studies were approved by the institutional IACUC. Inductionof animal models of proteinuria (n=4 rats/group) in WT rats aredescribed in previous publications in parenthesis: PAN (7), PHN (7), PANwith glucocorticoids (20), non-HIV collapsing glomerulopathy (18),nephrotoxic serum induced heterologous phase proteinuria (7); theforegoing references are hereby incorproated by reference for suchteachings. Anti-Thy1.1 nephritis was induced by injection of 200 mcg ofanti-Thy1.1 (Ox-7 hybridoma) or control IgG IV into different groups ofmale Wistar rats (100-125 gm, n=4 rats/group), and rats euthanized after24 and 72 hours.

The following techniques are described in prior publications: Taqmanreal time PCR (26), confocal imaging (7), in situ hybridization (27),promoter—reporter studies (7), immunogold EM (26), glomerular extractionand processing for Western blot (26), assessment of charge by PEI method(28); the foregoing references are hereby incorproated by reference forsuch teachings. For alcian blue staining, the pH of the stainingsolution was adjusted to 2.5 using acetic acid, and 0.1% nuclear fastred solution was used as a counterstain. Densitometry of glomerularbasement membrane alcian blue stain (20 glomeruli/rat, 3 rats/group) wasassessed using Image-Pro software (Media Cybernetics, Inc., BethesdaMd., USA). Densitometry of 2D gel Western blots was assessed usingGel-Pro Analyzer software (Media Cybernetics, Inc.). Taqman real timePCR primers and probes are listed in Table 1. For in situ hybridization,the digoxigenin labeled probe for rat Angptl4 included bp 1 to 548 ofthe ORF.

To obtain samples for post heparin LPL activity, rats were injectedintravenously with 10 units/100 gm weight of porcine heparin 15 minutesprior to euthanasia, and activity measured using an assay from RoarBiomedical, Inc (New York N.Y.). Serum triglycerides were measured inthe fasting state.

Injection of NTS into Angptl4 −/− Mice

Angptl4 −/− mice were provided to Sander Kersten as a kind gift from EliLily Corporation (Indianapolis Ind. USA). The study protocol wasapproved by the Animal Studies Committee at Wageningen University.Eleven week old male Angptl4 −/− or +/+ mice (n=4 mice/group) wereinjected intravenously with 1.5 mg γ2-NTS or normal sheep serum (SigmaAldrich St. Louis Mo. USA), spot urine samples collected, at 48 hours,mice euthanized at 72 hours, plasma collected for biochemicalmeasurements, and kidneys preserved for histological analysis. Urinealbumin was assessed by ELISA (Bethyl laboratories, Montgomery Tex. USA)and urine creatinine measured by mass spectrometry. To assess for footprocess effacement, the mean width of foot processes was first measuredin control treated Angptl4 +/+ mouse transmission electron micrographs(10 equally spaced readings/loop, 3 loops/glomerulus, 3glomeruli/kidney, 3 kidneys/group). Effacement was described as an over2.5 fold increase in mean width. Total and effaced foot processes werecounted in NTS treated or control treated Angptl4 −/− mice.

Studies with Archived Human Samples

Immunostaining of archived human kidney biopsies (n=5 biopsies percondition) was conducted on samples obtained via IRB approved protocolsat the Instituto Nacional de Cardiologia, Mexico City. Control kidneybiopsies used for these studies were sex and age matched protocolpre-transplant biopsies. Archival human sera for 2D gel electrophoresisand Western blot (n=4 samples/condition) were obtained from a previouslypublished study (29).

Generation of Transgenic Rats:

NPHS2-Angptl4 TG rats were generated as follows: The vector pTRE-tightwas digested with Stul and EcoRI to remove the minimum CMV promoterbetween bp 278 and 324, the 5′ overhangs blunt ended with T4 DNApolymerase, and re-ligated to generate pTRE-tight MP (minus promoter).For podocyte specific expression, a rat Angptl4 cDNA construct(including the signal sequence) with a C-terminal V5 tag was placedupstream of a SV40 polyA tail. The human NPHS2 promoter was clonedupstream by PCR using a published human NPHS2 promoter construct astemplate (gi22652661) without DMSO to exclude a naturally occurring loopbetween bps 2343 and 2568 to improve expression.

Transgenic rats were generated by microinjection of the digested DNAconstructs into fertilized Sprague Dawley eggs (conducted at Universityof Michigan), implantation into pseudopregnant host Sprague Dawleyfemales, and the resulting offsprings were genotyped by routine PCR andTaqMan genomic DNA real time PCR strategy using construct specific andcontrol genomic prolactin primer and probe combinations. Two founderlines for podocyte specific expression were generated. Data fromNPHS2-Angptl4 TG rat line 740 (5 copies of the transgene) and werestable over 4 generations, are presented. Urinary total protein wasassessed using the Bradford method (Biorad laboratories, Hercules Calif.USA), and albuminuria by ELISA (Bethyl laboratories, Montgomery Tex.USA).

Measurement of Rat Blood Pressure

Blood pressure and pulse rate were measured in six 5 month old wild typeand proteinuric heterozygous NPHS2-Angptl4 TG rats by the tail cuffmethod using the SC-1000 apparatus from Hetteras Instruments, Inc. (CaryN.C. USA). A minimum of 80 reading were analyzed per group.

Development and Characterization of a Stable Cell Line and RecombinantAngptl4 Protein

A stable cell line was developed using a rat Angptl4 pcDNA 3.1-V5/Hisconstruct, along with control empty vector stable cell lines. TheAngptl4 stable cell line develops 55,253±5,155 fold rat Angptl4 mRNAupregulation (over undetectable baseline as reference value of 1) andsecretes a 70 kDa protein in serum free conditions and a 55 kDa proteinin the presence of serum. The full length V5-His tagged protein that wasaffinity purified from serum free media using a Nickel affinity column.Western blot studies performed on 2D gels revealed that most of therecombinant Angptl4 migrated as a high isoelectric point (8.3-8.5)protein, and was non-phosphorylated.

Promoter—Reporter Studies.

The mouse Angptl4 promoter previously published (31) had atranscriptional start site at −183 bp upstream of ATG in the liver. Weconducted 5′ RACE and primer extension analysis using mouse kidney mRNAand confirmed the same transcriptional start site in the kidney. Next, a2 Kb fragment upstream of the transcriptional start site was clone usingBAC clone RP23-27D5 as a template into pSEAP2 basic. A 2 Kb humanAngptl4 promoter constructs were similarly generated using BAC cloneRP11-886P16.

Incubation of HEK 293 cells and GECs transfected with Angptl4-pSEAP orempty vector promoter—reporter constructs for mouse Angptl4 withdexamethasone (10⁻³ M) show a decline in SEAP activity at 48 hours. Thisdecline in activity is mediated via the glucocorticoid receptor, sinceit is reversed by the receptor antagonist mifepristone (2×10⁻⁵ M).Glucocorticoid sensitivity of the human promoter was also assessed(dexamethasone 10⁻⁴ M). All transfection reactions were normalized usingthe pβgal vector (Clontech, Mountain View Calif. USA).

In vitro and in vivo Studies with ManNAc

To study the effect of manNAc on the sialylation of recombinant protein,the Angptl4-HEK293 stable or pcDNA3.1-HEK293 control stable cell lineswere grown to confluence in 15 cm dishes, washed twice with warm PBS,then incubated with serum free DMEM without phenol red with or without25 mM manNAc for 48 hours, after which the media was harvested,concentrated, protein assay conducted, and loaded on the 2D gels (200μg/gel). Western blots of glomeruli or recombinant protein wereconducted using rabbit anti-Angptl4 (full length) antibody, preimmunerabbit serum and lectins SNA I-HRP and MAA-HRP (E-Y laboratories, SanMateo Calif. USA).

Subsequent in vitro studies with neutral pI Angptl4 (ManNAc treated) orhigh pI Angptl4 (untreated) used either His tag purified protein orconcentrated supernatant harvested from Angptl4-HEK293 stable in a samemanner Control protein was concentrated from control stable cell linesupernatant.

Pilot studies in heavily albuminuric NPHS2-Angptl4 rats revealed a largeManNAc dose requirement (8 mg/ml tap water) to maintain 20-30% reductionin albuminuria. To make the study affordable, 7 homozygous maleNPHS2-Angptl4 rats, age 3-3.5 months, with 15-25 fold higher albuminuriathan wild type rats, were given ManNAc 1 mg/ml of tap water for 12 days(ManNAc phase), after which three were euthanized and the othersreturned to plain tap water for another 24 days (washout phase). 18 hoururine collections were done twice (Day- 12 and Day 0) before the studyto confirm rising albuminuria, and periodically during the study.Another seven male NPHS2-Angptl4 rats of similar age were assessed forbaseline albuminuria levels, given normal tap water (control group),three euthanized on Day 12 and others followed up to day 36, after whichalbuminuria was reassessed and the animals euthanized. Daily waterintake of each rat was charted. At each euthanasia time point, kidneyswere removed after perfusion, glomeruli isolated and processed for 2Dgel electrophoresis. Albuminuria on Day 0 for each rats was denoted as100% and all subsequent albuminuria values were expressed relative toDay 0.

Statistical Analysis

Analysis of difference in proteinuria or gene expression involving threeor more groups was conducted by ANOVA with post analysis testing usingGraphPad InStat software, Version 3.05. For comparison of two groups,the unpaired Students t test in Microsoft Excel 2003 was used.

The foregoing description illustrates and describes the methods andother teachings of the present disclosure. Additionally, the disclosureshows and describes only certain embodiments of the methods and otherteachings disclosed, but, as mentioned above, it is to be understoodthat the teachings of the present disclosure are capable of use invarious other combinations, modifications, and environments and iscapable of changes or modifications within the scope of the teachings asexpressed herein, commensurate with the skill and/or knowledge of aperson having ordinary skill in the relevant art. The embodimentsdescribed hereinabove are further intended to explain best modes knownof practicing the methods and other teachings of the present disclosureand to enable others skilled in the art to utilize the teachings of thepresent disclosure in such, or other, embodiments and with the variousmodifications required by the particular applications or uses.Accordingly, the methods and other teachings of the present disclosureare not intended to limit the exact embodiments and examples disclosedherein. All references cited herein are incorporated by reference as iffully set forth in this disclosure.

TABLE 1 List of primers and probes used for Taqman real time PCRGene / transgene Forward primer Reverse primer Taqman probe Angpt14tctgggatctccaccatttttg tcaccgtccagcctccat caactgtgagatgacttc Angpt14cgccacccgcttacaca cagaggctggatctggaaaagt tgccaggaactcttt NPHS2-Angpt14tacaggctaccaccctgttgatc aaccgcgggccctctag ccatggaggctacagca constructProlactin cttgaagggattgaaaagataattagc ccatgagtcagaaaagcattgaacaggtgagcattttcctg (genomic)

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What is claimed:
 1. A method for determining the status of a subjectwith respect to a hyposialylated form of an Angptl4 polypeptide, saidmethod comprising the steps of: a. obtaining a sample containing theAngptl4 polypeptide from the subject; and b. determining a level ofsialylation of the Angptl4 polypeptide in the sample.
 2. The method ofclaim 1, wherein prior to determining the level of sialylation of theAngptl4 polypeptide, the Angptl4 polypeptide in the sample is separatedby isoelectric point.
 3. The method of claim 2, wherein the determiningstep is made on a portion of the Angptl4 polypeptide having anisoelectric point of 8.0 to 8.5.
 4. The method of claim 1, wherein thehyposialylated form of Angptl4 is characteristic of nephrotic syndromeor a diabetic condition.
 5. The method of claim 1 further comprisingcomparing the level of sialylation of the Angptl4 polypeptide from thesubject to a control value.
 6. A method for determining the status of asubject with respect to a hyposialylated form of an Angptl4 polypeptide,said method comprising the steps of: a. obtaining a sample containing anAngptl4 polypeptide from the subject; b. separating the Angptl4polypeptide in the sample by isoelectric point; and c. determining thelevel of sialylation of the Angptl4 polypeptide in the sample from aportion of the Angplt4 polypeptide having an isoelectric point of 8.0 to8.5.
 7. The method of claim 7, wherein the presence of a hyposialylatedform of Angptl4 is characteristic of nephrotic syndrome or a diabeticcondition.
 8. The method of claim 7 further comprising the level ofsialylation of the Angptl4 polypeptide from the subject to a controlvalue.
 9. The method of claim 9, wherein the control value is areference standard.
 10. A method for determining the efficacy of atreatment in a subject to increase the sialylation of an Angptl4polypeptide in the subject, said method comprising the steps of: a.obtaining a sample containing the Angptl4 polypeptide from the subject;b. determining the level of sialylation of the Angptl4 polypeptide inthe sample; and c. determining that the treatment is efficacious if thelevel of sialylation of the Angptl4 polypeptide from the sample is equalto or approaching the level of sialylation in a control value or if thelevel of sialylation of the Angptl4 polypeptide from the sample isincreased as compared to the level of sialylation of the Angptl4polypeptide from the subject determined prior to initiating saidtreatment.
 11. The method of claim 10, wherein prior to determining thelevel of sialylation of the Angptl4 polypeptide, the Angptl4 polypeptidein the sample is separated by isoelectric point.
 12. The method of claim12, wherein the determining step is made on a portion of the Angptl4polypeptide having an isoelectric point of 8.0 to 8.5.
 13. The method ofclaim 10, wherein the control value is a reference standard.
 14. Amethod for determining the efficacy of a treatment in a subject toincrease the sialylation of an Angptl4 polypeptide in the subject, saidmethod comprising the steps of: a. obtaining a sample containing theAngptl4 polypeptide from the subject; b. separating the Angptl4polypeptide in the sample by isoelectric point; c. determining the levelof sialylation of the Angptl4 polypeptide in the sample from a portionof the Angptl4 polypeptide having an isoelectric point of 8.0 to 8.5;and d. determining that the treatment is efficacious if the level ofsialylation of the Angptl4 polypeptide from the sample is equal to orapproaching the level of sialylation in a control value or if the levelof sialylation of the Angptl4 polypeptide from the sample is increasedas compared to the level of sialylation of the Angptl4 polypeptide fromthe subject determined prior to initiating said treatment.
 15. Themethod of claim 14, wherein the control value is a reference standard.16. A method for identifying a compound effective for increasing a levelof sialylation in an Angptl4 polypeptide, the method comprising thesteps of: a. providing an assay system that expresses the Angptl4polypeptide; b. introducing into the assay system a test compound; c.determining the level of sialylation of the Angptl4 polypeptide in thepresence of and in the absence of the test compound, wherein an increasein the level of sialylation of the Angplt4 polypeptide in the presenceof the test compound indicates the test compound is effective.
 17. Themethod of claim 16, wherein the Angptl4 polypeptide is a full lengthAngptl4 polypeptide, a biologically active fragment of the Angptl4polypeptide or a fusion protein which includes all or a portion of theAngptl4 polypeptide.
 18. The method of claim 16, wherein the assaysystem involves the use of a whole cell.
 19. The method of claim 19,wherein the cell naturally expresses the Angptl4 polypeptide or ismodified to express the Angptl4 polypeptide.
 20. The method of claim 16,wherein the level of sialylation is determined on a portion of theAngptl4 polypeptide having an isoelectric point of 8.0 to 8.5.